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Results for “stalled replication fork”

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At least 271 records · Page 15Linked to original sources
PubMed2005

Ionizing radiation-dependent gamma-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related.

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of gamma-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.

Arabidopsis↗
PubMed2000

Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest.

Individuals affected by the autosomal recessive disorder Werner's syndrome (WS) develop many of the symptoms characteristic of premature ageing. Primary fibroblasts cultured from WS patients exhibit karyotypic abnormalities and a reduced replicative life span. The WRN gene encodes a 3'-5' DNA helicase, and is a member of the RecQ family, which also includes the product of the Bloom's syndrome gene (BLM). In this work, we show that WRN promotes the ATP-dependent translocation of Holliday junctions, an activity that is also exhibited by BLM. In cells arrested in S-phase with hydroxyurea, WRN localizes to discrete nuclear foci that coincide with those formed by the single-stranded DNA binding protein replication protein A. These results are consistent with a model in which WRN prevents aberrant recombination events at sites of stalled replication forks by dissociating recombination intermediates.

Adenosine Triphosphatases↗
PubMed2003

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells.

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.

Animals↗
PubMed2002

Overlapping roles of the spindle assembly and DNA damage checkpoints in the cell-cycle response to altered chromosomes in Saccharomyces cerevisiae.

The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Delta mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase delta. Thus the specificity of this checkpoint may be more limited than previously recognized.

Calcium-Binding Proteins↗
PubMed2000

Homologous recombination as a mechanism for genome rearrangements: environmental and genetic effects.

Novel findings over the last 2 years have led to an increased emphasis on homologous recombination (HR) as both a pathway for DNA repair and a cause for genomic rearrangements. Indeed, environmental carcinogens increase the frequency of HR, as can be observed when two copies of a duplicated sequence recombine to delete the intervening sequences. Such HR events between dispersed homologous sequences may result in not only deletions, but also gene duplications or translocations. These types of genomic rearrangement have been observed to be the cause of several different genetic diseases, including cancer. In reflection of this, several genes have been identified that, when mutant, predispose an individual to an increased frequency of cancer. These genes have been shown to be either directly or indirectly involved in HR. In addition, HR is induced by a wide variety of carcinogens, preferentially in proliferating cells. This fits the most current models of recombination and its involvement in reinitiating stalled replication forks. Thus, 'correct' HR repair may act with high fidelity, an important issue for proliferating cells, but in the context of alternative homologous partner sequences, 'aberrant' HR can cause genomic rearrangements with dire consequences.

Animals↗
PubMed2004

Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways.

Fanconi anaemia (FA) is a chromosomal instability disorder characterized by cellular sensitivity to DNA interstrand crosslinking agents and a high risk of cancer. Six of the eight proteins encoded by the known FA genes form a nuclear complex which is required for the monoubiquitination of the FANCD2 protein. FANCD2 complexes and colocalizes with BRCA1, but its presumptive role in DNA repair has not yet been clearly defined. We used yeast two-hybrid analysis to test for interaction between FANCD2 and 10 proteins involved in homologous recombination repair. FANCD2 did not interact with RAD51, the five RAD51 paralogs, RAD52, RAD54 or DMC1. However, it bound to a highly conserved C-terminal site in BRCA2 that also binds FANCG/XRCC9. FANCD2 and BRCA2 can be coimmunoprecipitated from cell extracts of both human and Chinese hamster wild-type cells, thus confirming that the interaction occurs in vivo. Formation of nuclear foci of FANCD2 was normal in the BRCA2 mutant CAPAN-1 cells, which indicates that the recruitment of FANCD2 to sites of DNA-repair is independent of wild-type BRCA2 function. FANCD2 colocalized with RAD51 in foci following treatment with mitomycin C or hydroxyurea, and colocalized very tightly with PCNA after treatment with hydroxyurea. These findings suggest that FANCD2 may have a role in the cellular response to stalled replication forks or in the repair of replication-associated double-strand breaks, irrespective of the type of primary DNA lesion.

Animals↗
PubMed2000

Characterisation of the catalytically active form of RecG helicase.

Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery. However, several mechanisms to overcome such problems are beginning to emerge from studies with Escherichia coli. An important enzyme in one or more of these mechanisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/or bypass of the original lesion. To begin to understand how RecG might catalyse regression of fork structures, we have analysed what the catalytically active form of the enzyme may be. We have found that RecG exists as a monomer in solution as measured by gel filtration but when bound to junction DNA the enzyme forms two distinct protein-DNA complexes that contain one and two protein molecules. However, mutant inhibition studies failed to provide any evidence that RecG acts as a multimer in vitro. Additionally, there was no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP. These data suggest that RecG functions as a monomer to unwind junction DNA, which supports an 'inchworm' rather than an 'active rolling' mechanism of DNA unwinding. The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target and correlates with the very low abundance of replication forks within an E.COLI: cell, even during rapid growth.

Amino Acid Sequence↗
PubMed2000

Human 100-kDa homologous DNA-pairing protein is the splicing factor PSF and promotes DNA strand invasion.

Proteins promoting homologous pairing could be involved in various fundamental biological processes. Previously we detected two mammalian nuclear proteins of 100 and 75 kDa able to promote homologous DNA pairing. Here we report isolation and characterisation of the human (h) 100-kDa DNA-pairing protein, hPOMp100, from HeLa nuclei. The peptide sequences of hPOMp100 revealed identity to the human splicing factor PSF and a DNA-binding subunit of p100/p52 heterodimer of unknown function. Bacterially expressed PSF promotes DNA pairing identical to that of hPOMp100. hPOMp100/PSF binds not only RNA but also both single-stranded (ss) and double-stranded (ds) DNA and facilitates the renaturation of complementary ssDNAs. More important, the protein promotes the incorporation of a ss oligonucleotide into a homologous superhelical dsDNA, D-loop formation. A D-loop is the first heteroduplex DNA intermediate generated between recombining DNA molecules. Moreover, this reaction could be implicated in re-establishing stalled replication forks. Consistent with this hypothesis, DNA-pairing activity of hPOMp100/PSF is associated with cellular proliferation. Significantly, phosphorylation of hPOMp100/PSF by protein kinase C inhibits its binding to RNA but stimulates its binding to DNA and D-loop formation and may represent a regulatory mechanism to direct this multifunctional protein to DNA metabolic pathways.

Base Pairing↗
PubMed2004

Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.

Homologous recombination plays a key role in the restart of stalled replication forks and in the generation of genetic diversity. During this process, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction. In the presence of metal ions, the Holliday junction folds into the stacked-X structure that has two alternative conformers. Experiments have revealed the spontaneous transitions between these conformers, but their detailed pathways are not known. Here, we report a series of molecular dynamics simulations of the Holliday junction at physiological and elevated (400 K) temperatures. The simulations reveal new tetrahedral intermediates and suggest a schematic framework for conformer transitions. The tetrahedral intermediates bear resemblance to the junction conformation in complex with a junction-resolving enzyme, T7 endonuclease I, and indeed, one intermediate forms a stable complex with the enzyme as demonstrated in one simulation. We also describe free energy minima for various states of the Holliday junction system, which arise during conformer transitions. The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition.

Computational Biology↗
PubMed2004

REV1 accumulates in DNA damage-induced nuclear foci in human cells and is implicated in mutagenesis by benzo[a]pyrenediolepoxide.

The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2005

Torque-limited RecA polymerization on dsDNA.

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA-DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 +/- 0.7 k(b)T. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.

Biopolymers↗
PubMed2005

Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks.

Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.

Alkylating Agents↗
PubMed2002

Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype.

The clinical phenotype of Werner Syndrome (WRN) includes features reminiscent of accelerated aging and an increased incidence of sarcomas and other tumors of mesenchymal origin. This syndrome results from mutations in the WRN DNA helicase/exonuclease gene. We found that WRN deficient primary fibroblasts, as well as lymphoblastoid cell lines (LCLs), show reduced proliferative survival in response to 4-nitroquinoline-N-oxide (4NQO) and 8-methoxypsoralen (8MOP), compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand crosslinks, but not 8MOP mono-adducts, produced S-phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S-phase apoptosis in WRN-deficient diploid fibroblasts, in which drug hypersensitivity was entirely due to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S-phase may promote genomic instability and could help clarify the increased risk of sarcoma in WRN patients. Because interstrand crosslinks are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks.

4-Nitroquinoline-1-oxide↗
PubMed2004

Recombination and chromosome segregation.

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.

Adenosine Triphosphatases↗
PubMed2003

SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase.

Eukaryotic cells respond to DNA damage and stalled replication forks by activating protein kinase-mediated signaling pathways that promote cell cycle arrest and DNA repair. A central target of the cell cycle arrest program is the Cdc25A protein phosphatase. Cdc25A is required for S-phase entry and dephosphorylates tyrosine-15 phosphorylated Cdk1 (Cdc2) and Cdk2, positive regulators of cell division. Cdc25A is unstable during S-phase and is degraded through the ubiquitin-proteasome pathway, but its turnover is enhanced in response to DNA damage. Although basal and DNA-damage-induced turnover depends on the ATM-Chk2 and ATR-Chk1 pathways, how these kinases engage the ubiquitin ligase machinery is unknown. Here, we demonstrate a requirement for SCFbeta-TRCP in Cdc25A turnover during an unperturbed cell cycle and in response to DNA damage. Depletion of beta-TRCP stabilizes Cdc25A, leading to hyperactive Cdk2 activity. SCFbeta-TRCP promotes Chk1-dependent Cdc25A ubiquitination in vitro, and this involves serine 76, a known Chk1 phosphorylation site. However, recognition of Cdc25A by beta-TRCP occurs via a noncanonical phosphodegron in Cdc25A containing phosphoserine 79 and phosphoserine 82, sites that are not targeted by Chk1. These data indicate that Cdc25A turnover is more complex than previously appreciated and suggest roles for an additional kinase(s) in Chk1-dependent Cdc25A turnover.

Amino Acid Motifs↗
PubMed2005

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast.

The DNA replication checkpoint is a complex signal transduction pathway, present in all eukaryotic cells, that functions to maintain genomic integrity and cell viability when DNA replication is perturbed. In Schizosaccharomyces pombe the major effector of the replication checkpoint is the protein kinase Cds1. Activation of Cds1 is known to require the upstream kinase Rad3 and the mediator Mrc1, but the biochemical mechanism of activation is not well understood. We report that the replication checkpoint is activated in two stages. In the first stage, Mrc1 recruits Cds1 to stalled replication forks by interactions between the FHA domain of Cds1 and specific phosphorylated Rad3 consensus sites in Mrc1. Cds1 is then primed for activation by Rad3-dependent phosphorylation. In the second stage, primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation. This two-stage activation mechanism for the replication checkpoint allows for rapid activation with a high signal-to-noise ratio.

Catalytic Domain↗
PubMed2002

ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase.

The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks.

Cell Cycle Proteins↗